Mineral precipitation methods

ABSTRACT

The present invention provides methods for mineral precipitation of porous particulate starting materials using isolated urease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15/029,316 filed on Apr. 14, 2016, now U.S. Pat. No. 10,792,367 entitled “MINERAL PRECIPITATION METHODS.” U.S. Ser. No. 15/029,316 is a national stage entry of PCT/US2014/062540 filed on Oct. 28, 2014 and published on May 7, 2015 as WO/2015/065951 entitled “MINERAL PRECIPITATION METHODS.” PCT/US2014/062540 claims priority to, and the benefit of, U.S. Provisional Application No. 61/896,340 filed on Oct. 28, 2013. PCT/US2014/062540 also claims priority to, and the benefit of, U.S. Provisional Application No. 61/916,908 filed on Dec. 17, 2013. Each of the foregoing applications are incorporated herein by reference in their entirety for all purposes, except for any subject matter disclaimers or disavowals.

STATEMENT OF U.S. GOVERNMENT INTEREST

This invention was made with government support under grant number 1233658 awarded by the National Science Foundation and grant number 0856801 by the National Science Foundation. The government has certain rights in the invention.

SUMMARY

The present invention provides methods for mineral precipitation, comprising combining a porous, particulate starting material with

(a) isolated urease;

(b) urea; and

(c) a source of divalent cations;

wherein (a), (b), (and (c) are provided in amounts effective and the combining is carried out under conditions suitable to cause carbonate precipitation of the starting material. In another embodiment, the method further comprises introducing a clay slurry into the starting material prior to or concurrent with combining the starting material with the isolated urease, the urea, and the source of divalent cations.

In another aspect, the invention provides methods for mineral precipitation, comprising

(a) combining a porous starting material with a clay slurry to form a starting material complex; and

(b) combining the starting material complex with

-   -   (i) isolated urease;     -   (ii) urea; and     -   (iii) a source of divalent cations;

wherein (i), (ii), and (iii) are provided in amounts effective and the combining is carried out under conditions suitable to cause carbonate precipitation of the starting material complex.

In one embodiment of either aspect of the invention, the methods can be used for one or more of improving bearing capacity of foundations; stabilizing slopes, reducing settlement potential of foundations or embankments; reducing the potential for earthquake-induced liquefaction; mitigating the potential for damaging ground displacements subsequent to earthquake-induced liquefaction; increasing lateral resistance of foundations; enhancing stability of slopes or embankments; reducing lateral earth pressures on retaining walls; increasing passive resistance of retaining walls; increasing capacity of ground anchors or soil nails; increasing the side resistance and tip resistance of deep foundations; facilitating tunneling in running or flowing ground; stabilizing excavations bottoms; soil erosion control; and groundwater control. In one embodiment, the starting material is saturated. In another embodiment, the starting material is selected from the group consisting of unconsolidated sand, silt, clay, other sediments, and sawdust.

DESCRIPTION OF THE FIGURES

FIG. 1. LV-Scanning electron microscope images of a) well-grown and cementing calcite crystals; b) cementing calcite crystals at inter-particle contact; c) indention of quartz surface (arrows) and nucleation of calcite crystals (red arrows); d) calcite crystal growing on quartz surface.

FIG. 2. P-q plot failure envelopes for 20-30 silica sand: ▪Cemented (D_(r)=60%); ∘Uncemented (D_(r)=60%).

FIG. 3. P-q plot failure envelopes for F-60 silica sand: ▪Cemented (D_(r)=35%); ∘Uncemented (D_(r)=37%).

FIG. 4. Results of triaxial compression tests performed using F-60 Ottawa sand (medium sand).

FIG. 5. Results of triaxial compression tests performed using medium sand w/bentonite slurry.

FIG. 6. Results of triaxial compression tests performed using F-60 fine sand.

FIG. 7. Image from mix & compact columns using silica 20-30. These are silica sand particles variously covered with CaCO₃ and cemented at the inter-particle contacts.

FIG. 8. Image from same mix & compact columns using silica 20-30 as shown in FIG. 7. Note the concave CaCO₃ cement where a sand particle was bonded.

FIG. 9. Image from the same mix & compact columns using silica 20-30 as shown in FIG. 7. These are silica sand particles covered with a growing CaCO₃ layer that forms cement at the inter-particle contact. The large particles to the left appear to be well-developed CaCO and possibly evaporates (far left).

FIG. 10. Low-resolution image from a 4″×12″ PVC column #2 using silica 20-30 with bentonite; note the scale (10 μm). These are growing CaCO₃ crystals covering silica sand particles (not seen). The CaCO₃ is randomly covered or associated with clumps of bentonite, which may be serving as points of nucleation.

FIG. 11. Image from the 4″×12″ PVC column #1 using silica 20-30. These are growing CaCO₃ crystals covering silica sand particles. The relatively smooth concave feature is an inter-particle cementation contact.

FIG. 12. Image from the same 4″×12″ PVC column #1 using silica 20-30 as FIG. 11, but zoomed-in to the relatively smooth concave inter-particle feature described above; these are CaCO₃ crystals covering silica sand particles.

FIG. 13. Image from the same 4″×12″ PVC column #1 using silica 20-30 as in FIG. 11. These are CaCO₃ crystals covering silica sand particles.

FIG. 14. Image from the same 4″×12″ PVC column #1 using silica 20-30 as in FIG. 11.

FIG. 15. Image from the 4″×12″ PVC column #3 using silica F-60. These are silica sand particles variously covered with CaCO₃ crystals.

FIG. 16. Image from the same 4″×12″ PVC column #3 using silica F-60 as in FIG. 15, but at higher magnification. Note the inter-particle cementation.

FIG. 17. Image from the same 4″×12″ PVC column #3 using silica F-60 as in FIG. 15, but showing different location. These are silica sand particles covered with CaCO₃ crystals.

FIG. 18. Image from the same 4″×12″ PVC column #3 using silica F-60 as in FIG. 15, but at higher magnification. These are silica sand particles covered with CaCO₃ crystals.

FIG. 19. Image from the same 4″×12″ PVC column #3 using silica F-60 as in FIG. 15, but at lower magnification. These are silica sand particles covered with CaCO₃ crystals.

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.

All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides mineral precipitation and/or host medium cementation methods, comprising combining a porous particulate starting material with

(a) isolated urease;

(b) urea; and

(c) a source of divalent cations;

wherein (a), (b), and (c) are provided in amounts effective and the combining is carried out under conditions suitable to cause carbonate precipitation of the starting material. The starting material may be either saturated or unsaturated; in a preferred embodiment the starting material is saturated.

Induced carbonate precipitation can enhance the stiffness, strength, and liquefaction resistance of soil. Methods currently under investigation for soil improvement by inducing carbonate precipitation microbially are restricted to fine-grained or coarser sands, are limited by the need to stimulate microbial growth either in the subsurface or ex situ in a reactor vessel and by plugging of the pores by carbonate precipitation and the microbial mass. The methods of the present invention provide significant advantages over prior methods, by permitting precipitation in permeable materials under anaerobic conditions, e.g. in a saturated porous starting material, and by facilitating precipitation in finer grained soils such fine sands and silts, than was previously possible. Furthermore, the methods of the invention mitigate plugging issues that plague prior methods. The methods provide an alternative to commonly used soil improvement techniques such as deep soil mixing, stone columns, penetration and compaction grouting, and rammed aggregate piers.

The methods can be used, for example, in improving the bearing capacity of foundations, reducing settlement potential of foundations and embankments, increasing the lateral resistance of foundations, reducing the potential for earthquake-induced liquefaction; mitigating the potential for damaging ground displacements subsequent to earthquake-induced liquefaction; enhancing the stability of slopes and embankments, reducing lateral earth pressures on retaining walls, increasing the passive resistance of retaining walls, increasing the capacity of ground anchors and soil nails, increasing the side resistance and tip resistance of deep foundations, facilitating tunneling in running or flowing ground (dry or saturated cohesionless soil), stabilizing the bottom of excavations, soil erosion control, groundwater control.

“Carbonate cementation” means mineral precipitates that may include one or more cations such as calcium, magnesium, iron and others that may produce one of several phases of carbonate minerals, including but not limited to calcite. In a preferred embodiment, calcium carbonate precipitates form cementation bonds at inter-particle contacts in the particulate starting material and fill in void spaces in the porous starting materials (thereby increasing the tendency of the starting material to dilate, or expand in volume, when sheared), and/or cementation of adjacent particles of the starting material.

As used herein, “saturated” means that the soil has reached its maximum water content; if any more water is added it will either drain downward, flow upwards, or turn the soil into a suspension wherein there is little to no inter-particle contact. In one example, the saturated starting material is below the water table. When the starting material is below the water table it is usually saturated, unless there is gas in the soil. Saturated zones may also exist above the water table due to capillary rise, with the extent of the saturated zone above the water table depending upon the pore size of the starting material.

As used herein, “isolated urease” is urease that is extracted from cells and cellular materials. The urease may be synthetically produced or obtained by extraction from any suitable source, including but not limited to bacteria, plants, invertebrates, and fungi. In one non-limiting embodiment, a plant derived urease extract can be used. For example, urease activity is present in various plant leaves and this activity can be realized using crude extracts of the leaves, or isolated enzyme. Urease enzyme as discussed herein is characterized by the reaction it catalyzes and identified by EC 3.5.1.5 (i.e. Reaction: urea+H₂O═CO₂+2NH₃). In one embodiment, the urease enzyme is isolated from the jack-bean plant (SEQ ID NO: 1). The amino acid sequences of exemplary ureases for use with the present invention are provided below. However, it will be clear to those of skill in the art that any enzyme identified by EC 3.5.1.5 can be used in the methods of the invention, including but not limited to a urease comprising or consisting of any one of SEQ ID NOS: 2-5, where SEQ ID NO:2 is a soybean urease, SEQ ID NO:3 is a Agaricus bisporus urease, SEQ ID NO:4 is a Schizosaccharomyces pombe (strain 972/ATCC 24843) urease, SEQ ID NO:5 is a Sporosarcina pasteurii urease, and SEQ ID NO:6 is a Pseudomonas syringae (strain B728a) urease.

The appropriate amount of urease needed can be determined by one of skill in the art based on the teachings herein; factors to be considered in determining an appropriate amount of urease include, but are not limited to:

(a) urease source type (e.g. Jack bean vs. other source)

(b) urease purity, which dictates enzymatic activity (i.e. rate of conversion of urea to products); and

(c) the stability/half-life of the enzyme matrix used, where the “enzyme matrix” refers to the specific form of the enzyme mixture used such as liquid, powder and/or solid when combined with or used apart from stabilizers, buffers, fillers or other media to facilitate its desired use.

For example, assuming that for practical purposes that transport via diffusion, advection and dispersion is not limiting the availability of urea or calcium to the enzymes—or vice versa—(e.g. we thoroughly mix the soil and cementation constituents or we actively pump the cementation constituents into the soil or do something to assure that the right constituents get to where they need to be), then in a homogenous soil (i.e. without zones of blocked flow or disproportionately high/preferential flow) we could expect an approximately linear relationship between urea conversion and required amount(s) of enzyme needed to convert “x” grams of urea to “y” grams of calcium carbonate over a given time frame. This is also dependent on the on the amount of calcium ions available for precipitation. A sufficiently high concentration of calcium to form calcium carbonate is needed, with hydrolysis of urea just one part of the overall process. If a soil mass requires a total amount “x” grams of urea to be converted into products for calcium carbonate formation, and “y” grams of enzyme can only convert 50% of “x” during its functional life time, then theoretically twice as much enzyme is needed to fully convert “x” grams of urea.

Urea is an organic compound of the chemical formula CO(NH₂)₂. Urea is a colorless, odorless, highly water soluble substance with very low toxicity (LD50=12 g/kg for mouse, Agrium MSDS), and is widely commercially available. Any suitable source of urea can be used, including but not limited to those disclosed herein.

The appropriate amount of urea can be determined by one of skill in the art based on the teachings herein; factors to be considered in determining an appropriate amount of urea include, but are not limited, to the amount of carbonate required for the particular application as determined on a stoichiometric basis. As will be understood by those of skill in the art, the amount of carbonate precipitation required varies from one use to another.

Several divalent cations, primarily alkaline earth metals (including but not limited to calcium and magnesium), that satisfy the crystalline structure constraints of calcite or calcium mineral carbonates can be used to precipitate carbonate minerals in the present methods. Any suitable source of divalent cations can be used, including but not limited to salts of organic and inorganic compounds such as nitrates, nitrites, chlorides, sulfates, oxides, acetates, silicates, oxalates or mixtures thereof.

The appropriate amount of ions can be determined by one of skill in the art based on the teachings herein; factors to be considered in determining an appropriate amount of ions include, but are not limited the required amount of carbonate precipitate required for the particular application as determined on a stoichiometric basis. In one non-limiting example, if 100 grams (approximately 1 mole) of calcium carbonate (CaCO₃) is desired, then 1 mole of urea ((NH₂)₂CO) and at least 1 mole of calcium (Ca²⁺) are required (the urea also provides the necessary 1 mole of carbon).

Any suitable starting material, or combinations of starting materials, may be used in the methods of the invention, such as those having a particulate structure or those consisting of discrete soil particles forming a stable framework (or skeleton) or relatively impervious blocks delineated by an interconnected network of fractures or fissures. In a preferred embodiment, the starting material may be unconsolidated or partially consolidated particulate material such as sand, silt, soil, clay, sediments, sawdust or other material that is amenable to in situ cementation, or combinations thereof. Such starting materials may be mixed in situ or mixed and compacted with the isolated urease, the urea, and the source of divalent cations. In a further preferred embodiment, the starting material is saturated.

In further embodiments, the starting material may be gravel, igneous, metamorphic, or sedimentary rocks including but not limited to conglomerate, breccia, sandstone, siltstone, shale, limestone, gypsum, and dolostone, or combinations thereof. In one preferred embodiment, the starting material comprises sand. In another preferred embodiment, the starting material comprises silt. In a further preferred embodiment, the starting material is fractured crystalline rock or cracked concrete.

As used herein, “particulate” starting materials are starting materials comprising separate particles. The starting material is “porous” in that it enables sufficient passage of the isolated urease, the urea, and/or the source of calcium or other ions and constituents including, but not limited to, buffers and stabilizers, to enable carbonate precipitation with or without cementation. In some applications, the method may work simply by filling the void spaces (e.g. soil pores, rock fractures) with the carbonate precipitate, without any actual cementation of/bonding to the host material. The components can be combined in any way suitable in light of the specific starting material, the amount of starting material, the components to be used, etc. In various embodiments, the starting material and components are combined by a technique selected from the group consisting of flushing, injecting, mixing, spraying, dripping or trickling onto or into the starting material. The starting material may also be immersed in one or more ways as described above. In addition, secondary non-specific methods may be employed to facilitate carbonate precipitation including, but not limited to, moisture control measures, crystal seeding, and initiation of nucleation sites. In one embodiment, the methods comprise mixing powdered urease or urease in solution with a particulate starting material prior to percolation of a solution comprising the urea and the divalent ion source or combining the urease, urea, and divalent ion in solution with the starting material, mixing them with in situ or mixing them ex situ and then placing or compacting the mixture. In another embodiment, the combining comprises injection of the isolated urease, urea, and/or divalent cations into the starting material via one or more central porous injection tubes, such as are described in the examples that follow. Any number of such injection tubes may be used, depending on the size and depth of the starting material to be treated, among other factors, and any design or size of such injection tubes may be used. In one non-limiting embodiment, field injection tubes may comprise 2 inch or 3 inch diameter perforated pipe that is vibrated or pushed into the soil. The tubes may be placed at varying depths and orientations in the starting material. In one embodiment, injection occurs all along the length of the tube at once, or may comprise injection over small intervals (such as 1-3 foot intervals) working from one end of the tube, preferably from the bottom of the injection tube, up, which can be accomplished, for example, by sealing off sections where injection is to occur with packers, as in known in grouting technology.

It will be understood by those of skill in the art that the step of “combining” the starting material with effective amounts of isolated urease, urea, and ions covers any process that results in the bringing together of the three constituents in a manner that results in precipitation of carbonate minerals in the starting material. The reactants may be added to the starting material simultaneously or sequentially. For example, there may be applications where one or two of the constituents are already present in the starting material, in which case the step of “combining” will involve the addition of only the missing components. In one embodiment, the urea and ions are admixed and then added to the urease prior to application to the starting material. However, it will be appreciated by those of skill in the art that the constituents may be combined in other ways to carry out the method of the invention.

By manipulating the relative effective amounts of the various components, the methods of the present invention enable the user to control carbonate precipitation by controlling the amount of carbonate formed and the rate at which it is formed. This flexibility means the methods of the present invention can be used in a wide range of applications from those that require a reasonably modest increase in the strength, stiffness, or dilatancy or modest decrease in the permeability in the starting material to those that require larger changes in the relevant property. As used herein, “dilatancy” refers to the tendency of a soil to expand in volume during shear. This is an important property, for example, with respect to reducing liquefaction potential.

The effective amounts of the various reactants combined according to the method of the present invention may vary depending, at least, on the amount of urease used, the characteristics of the starting material and the conditions under which precipitation is to occur, the desired final strength, stiffness, dilatancy, or permeability of the treated porous material and the amounts of the other reactants in the reaction mix. The present application enables those of skill in the art to determine the relative amounts of the various reactants required for a given application and to apply the method to various starting materials and for a variety of end uses. The method of the present invention may be adapted to allow for the rate of mineral precipitation to be controlled, as required. When rapid or slow formation of the precipitate is desired the amounts and/or relative amounts of the reagents can be selected accordingly to bring about the desired rate of formation. In one non-limiting example, enhancement of the methods may comprise providing stronger nucleation sites on particles of the starting material by high-pH pretreatment of the particles of the starting material to be improved.

Depending on the requirements of a particular application or mode of use of the present invention, rapid formation of the precipitate may be required. Alternatively it may be preferred for the precipitate to be formed slowly. Based on the teachings herein, those of skill in the art will be able to modify the protocol to attain faster or slower formation of the precipitate.

The methods of the invention may be repeated (once, twice, three times, or more) in order to attain the desired amount of mineral precipitate strength, stiffness increase, or permeability reduction. When the method is repeated to gain incremental increases in strength or stiffness or reduction in permeability, not all of the reagents need to be added each time. For example, residual urease activity may still be sufficient for one or more subsequent rounds of the method. A skilled person is readily able to determine the particular amounts of reagents required for use in subsequent rounds of the method of the present invention.

The methods may be applied in situ without disturbing the starting material. This is particularly important for applications where the starting material is delicate or fragile or for other reasons must not be disturbed. Examples include, but are not limited to, when applied in the field where the soil to be improved (e.g. made resistant to earthquake-induced liquefaction) is underneath or adjacent to an existing structure or facility that is sensitive to ground movement (e.g. settlement or heave).

As will be understood by those of skill in the art, the methods may comprise use of other components as appropriate for a given use. In one embodiment, the methods may further comprise use of a stabilizer (including but not limited to powdered milk) to increase enzyme stability and functional time. The methods can be carried out under any temperature conditions suitable to promote carbonate cementation.

In another embodiment, the method further comprises introducing a clay slurry into the starting material prior to or concurrent with combining the starting material with the isolated urease, the urea, and the source of divalent cations. This embodiment is particularly useful when the starting material comprises a high permeability starting material (e.g. a coarse-grained soil), as it will help to retain the active components in the starting material where cementation is desired; the clay particles may also serve as nucleation points for carbonate precipitation. As used herein, a “clay slurry” is any clay in suspension. In various non-limiting embodiments, the clay may comprise montmorillonite clay (also known as bentonite), attapulgite, or combinations thereof. The amount of clay slurry, the specific amount of clay in the slurry, and the timing/number of times the clay slurry is administered may vary depending, at least, on the characteristics of the starting material and the conditions under which precipitation is to occur, the desired final strength, stiffness, dilatancy, or permeability of the treated porous material and the amounts of the other reactants in the reaction mix. The present application provides examples that enable those of skill in the art to determine the relative amounts of the clay slurry appropriate for a given application and to apply the method to various starting materials and for a variety of end uses.

In a preferred embodiment, the starting material comprises a column of starting material. As used herein, a “column” refers to relatively linear prisms of soil that are to be stiffened and/or strengthened to reinforce the uncemented soil mass and/or transfer load to greater depths in the soil stratum. As will be understood by those of skill in the art, the prism of soil extends below a surface of the starting material. In various embodiments, the column is at least 0.5 meters long and 0.1 meters in diameter. In various further embodiments, the column is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or more meters long/deep. In various further embodiments, the column is at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, or more meters in diameter. In one exemplary embodiment, for the mitigation of earthquake-induced liquefaction under residential structures, the columns are between about 1-foot to 5-feet in diameter; more preferably about 3 feet in diameter and up to 60 feet long. In this embodiment, the methods comprise combining the starting materials so that carbonate precipitation of the starting material occur at in a radial pattern around an injection tube (e.g. perforated pipe) to form the column. In one embodiment, carbonate precipitation occurs at only one or more specific areas within the column, such as at a specific location where the starting materials are combined within the column (for example, where liquefiable soil strata is intersected by the injection tube). In another embodiment, carbonate precipitation occurs throughout the column.

This embodiment provides further improvements over prior methods, which are focused on improvement of the entire mass of soil in the improvement zone. In this embodiment, the methods permit production of stone-like materials via local cementation. Columns of improved starting materials, such as soils, have broad application in geotechnical practice, including reducing the potential for earthquake-induced liquefaction; mitigating the potential for damaging ground displacements subsequent to earthquake-induced liquefaction, improvement of foundation bearing capacity, support of embankments, slope stabilization, stabilization of the base of excavations, support of underground openings, and a variety of other geotechnical purposes. The limitations associated with bio-plugging can also be mitigated by using this technique to improve columns of soil (rather than the entire soil mass). Such columns of improved starting material possess, for example, increased strength, stiffness, and liquefaction resistance relative to the non-treated starting material.

In another embodiment, the methods can be used for fugitive dust control (e.g., wind-blown erosion).

In one non-limiting example, consider a 4.2-meter long, 1-meter diameter column of soil with a porosity of 0.31 is to be improved by precipitating 5% calcium carbonate by weight. This column has a total volume of 3.29 m³ and a pore space of 1.0 m³. Assuming a calcium carbonate density of 2.71 g/cm³ (2710 kg/m³), approximately 136 kg of CaCO₃ is used to precipitate in the improvement zone (i.e. the column). To obtain 136 kg of CaCO₃ (1,360 moles), 81.6 kg of urea (1,360 moles) and 54.4 kg of calcium (1,360 moles) are used, assuming that all added constituents are available and used in the reaction process to precipitate CaCO₃. Since there are 2 amine groups per urea molecule, 2,720 moles of ammonia nitrogen and 1,360 moles of carbon dioxide are released from 81.6 kg of urea. Assuming a minimum urease activity of 15,000 units/gram urease powder (type 3 urease from Jack bean [Sigma Aldrich]), where one-unit is the release of 1.0 mg ammonia nitrogen from urea in 5 minutes at pH 7.0 at 20° C., then 10 grams of this particular grade of enzyme (150,000 units) will release 150 grams of ammonia nitrogen (≈8.8 moles) in 5 minutes. Therefore, 26 hours (1546 minutes) are required to fully catalyze the release of carbon dioxide and ammonia from 81.6 kg of urea.

Any suitable technique/configuration for introducing the components into the column may be used as deemed appropriate. For introduction of the urease into columns, injection could be done at one site per column or at multiple sites along the column. If multiple sites, then the concentration may the same or different at the different locations.

In a second aspect, the present invention provides kits comprising

(a) isolated urease;

(b) urea;

(c) a source of divalent cations, preferably calcium ions; and

(d) a clay slurry;

wherein (a), (b), (c) and (d) are provided in amounts effective to cause carbonate cementation when combined with a porous, particulate starting material.

All definitions, embodiments, and combinations thereof of the first aspect apply equally to this second embodiment, unless the context clearly dictates otherwise. Thus, the kits may further contain any of the components or combinations thereof disclosed for use with the methods of the invention, including but not limited to stabilizers, buffers, nucleation aids, etc.

The use of plant-derived urease enzyme offers many benefits over the use of microbially-generated urease to induce carbonate cementation for soil improvement, a process that has garnered much attention recently. In this biogeochemical soil improvement process, urea hydrolysis is catalyzed by the urease enzyme (urea amidohydrolase), a widely occurring protein found in many microorganisms, higher order plants, and some invertebrates, to precipitate calcium carbonate in the presence of calcium. The calcium carbonate precipitate (CaCO₃) forms cementation bonds at inter-particle contacts and also fills the void space in granular soils. Urease is a nickel-dependent, metalloenzyme that is approximately 12 nm by 12 nm. By comparison, nearly all known bacteria that generate urease are greater than 300 nm in diameter, with the majority in the range of 500-5000 nm. As soil improvement by ureolytic carbonate precipitation requires penetration of the pore spaces by the improvement media, the small size of the enzyme affords the use of plant-derived enzyme a distinct advantage over microbial methods, including the ability to penetrate finer grained soils and less sensitivity to bioplugging (clogging of the pore space by the precipitate). An additional benefit of the use of plant-derived enzyme compared to microbially-derived enzyme is that the 100% of the carbon in the substrate is available for conversion to CaCO₃. Furthermore, plant derived enzyme is widely available.

In another aspect, the invention provides mineral precipitation methods, comprising

(a) combining a porous, particulate starting material with a clay slurry to form a starting material complex; and

(b) combining the starting material complex with

-   -   (i) isolated urease;     -   (ii) urea; and     -   (iii) a source of divalent cations;

wherein (i), (ii), and (iii) are provided in amounts effective and the combining is carried out under conditions suitable to cause carbonate precipitation of the starting material complex.

All terms and embodiments disclosed above can be used in this aspect of the invention. This aspect of the invention may be used with any starting material (whether saturated or not), including but not limited to all starting materials disclosed herein. In one embodiment, the soil is non-saturated; in another the starting material is saturated.

The methods of this aspect of the invention are particularly useful when the starting material comprises a high permeability starting material (e.g. a coarse-grained soil), as it will help to retain the active components in the starting material where cementation is desired; the clay particles may also serve as nucleation points for carbonate precipitation. As used herein, a “clay slurry” is as defined above. In various non-limiting embodiments, the clay may comprise montmorillonite clay (also known as bentonite), attapulgite, or combinations thereof. The amount of clay slurry, the specific amount of clay in the slurry, and the timing/number of times the clay slurry is administered to generate the “starting material complex” may vary depending, at least, on the characteristics of the starting material and the conditions under which precipitation is to occur, the desired final strength, stiffness, dilatancy, or permeability of the treated porous material and the amounts of the other reactants in the reaction mix. The present application provides examples that enable those of skill in the art to determine the relative amounts of the clay slurry appropriate for a given application and to apply the method to various starting materials and for a variety of end uses.

In various non-limiting embodiments, the methods of this second aspect of the invention may comprise or further one or more of the following:

-   -   the starting material is a column of the starting material;     -   the methods are used for one or more of improving bearing         capacity of foundations; reducing settlement potential of         foundations or embankments; reducing the potential for         earthquake-induced liquefaction; mitigating the potential for         damaging ground displacements subsequent to earthquake-induced         liquefaction; increasing lateral resistance of foundations;         enhancing stability of slopes or embankments; reducing lateral         earth pressures on retaining walls; increasing passive         resistance of retaining walls; increasing capacity of ground         anchors or soil nails; increasing the side resistance and tip         resistance of deep foundations; facilitating tunneling in         running or flowing ground; stabilizing excavations bottoms; soil         erosion control; and groundwater control;     -   the starting material is selected from the group consisting of         sand, silt, clay, other sediments, sawdust, igneous rocks,         metamorphic rocks, gravel, fractured crystalline rocks, cracked         concrete and sedimentary rocks including but not limited to         conglomerate, breccia, sandstone, siltstone, shale, limestone,         gypsum, and dolostone, and combinations thereof;     -   the isolated urease comprises jack bean urease;     -   the source of divalent cations comprises a source of divalent         calcium ions;     -   the combining step is carried out more than once;     -   the combining comprises         -   (i) mixing the urease with the starting material; and         -   percolating or injecting a solution comprising the urea and             the source of divalent cations into the column;     -   the percolating or injecting is carried out two or more times;         and/or     -   the urease is mixed with starting material complex in only a         portion of the starting material complex prior to the         percolating or injecting step.

Example 1. Carbonate Cementation Via Plant Derived Urease Methods Ottawa 20-30 Sand

Laboratory column tests were conducted using plant derived urease to induce CaCO₃ precipitation in Ottawa 20-30 sand These tests were carried out in 6″×2″ (152 mm×51 mm) acrylic tubes and membrane-lined 2.8″×6″ (71 mm×152 mm) split molds (for creating specimens for triaxial testing). Three acrylic tubes and two columns for triaxial testing were filled with 20-30 Ottawa silica sand (mean grain size 0.6 mm, coefficient of uniformity 1.1) and treated as follows: tube #1: the sand was dry pluviated via funnel at ≈3″ (76 mm) drop height and then received 5 applications of a cementation solution containing urea and calcium chloride mixed with 1.4 g/L enzyme (total solution volume≈300 ml); tube #2: sand was added in same manner as tube #1 and then received 2 applications (≈150 ml total) of the same cementation solution mixed with 1.4 g/L enzyme; tube #3: the lower-third of tube was filled with sand and dry enzyme (≈3 g), the remainder of the tube contained dry pluviated sand without enzyme, and the tube then received 2 applications (≈150 ml) of the cementation fluid with no enzyme added.

Approximately 100 mL of a pH=7.8 solution containing 383 mM urea (reagent grade, Sigma-Aldrich), 272 mM CaCl₂-2H₂O (laboratory grade, Alfa Aesar) was used for the first application in each acrylic tube. Subsequent applications employed approximately 50 mL of a pH=7.6 solution containing 416 mM urea and 289 mM CaCl₂-2H₂O. Solution concentrations, while variable, were formulated within a reasonably similar range as a matter of convenience. In each application, the cementation fluid was poured into the top of the acrylic tube with the bottom closed off. The cementation fluid was allowed to stand, loosely covered, in the acrylic tube for at least 24 hours and then drained out the bottom of the cylinder. The next application followed immediately after drainage was complete. Drainage was accomplished by puncturing the base of the cylinder with a 20-gauge needle. When drainage was complete, the needle was removed and the puncture was plugged with a dab of silicone. Occasionally, the needle became plugged and an additional needle was inserted through the base. The triaxial columns were filled with sand in the same manner as tube 1 and then received 2 applications (each application 250 ml) of cementation solution with 1.4 g/L enzyme.

In each application of cementation fluid, the fluid was added until it rose to approximately ½-inch (12-mm) above the soil line. After 2 applications, tubes #2 and #3 were allowed to air dry for several days and then analyzed. Experimentation with tube #1 was continued for several more days as three more batches of cementation fluid were applied. The last 2 applications of cementation fluid were allowed to slowly drain through the needle in the base immediately after application rather than sit for 24 hours (drainage rate ≈10-25 ml/hour). The triaxial columns were allowed to stand for at least a week after the second cementation fluid application and then drained.

After drainage was complete, the triaxial columns were moved to a triaxial testing device. After draining the specimens from the acrylic tubes and after the completion of the triaxial tests, all samples were triple washed with de-ionized water. Tubes #2 and #3 were separated in 3 layers, while tube #1 was separated into six layers (for better resolution). Each layer from the specimens in the acrylic tubes and the entire mass of the triaxial specimens were acid washed to determine CaCO₃ content by oven drying for 48 hours, weighing, digesting with warm 1M HCl, washing, drying, and reweighing to determine carbonate mineral content.

Several of the cemented specimens were analyzed for mineral identification using X-Ray Diffraction (XRD). Samples were ground in an agate mortar and pestle and powdered onto a standard glass slide for analysis. Scanning electron microscopy (SEM) imaging was performed on intact cemented chunks of material with an Agilent 8500 Low-Voltage SEM (LV-SEM). A LV-SEM is a field emission scanning electron microscope capable of imaging insulating materials, such as organic and biological substances without the need for a metal coating and without causing radiation damage to samples.

Ottawa F-60 Sand

A triaxial column was prepared using Ottawa F-60 silica sand (mean grain size 0.275 mm, coefficient of uniformity 1.74) to investigate enzymatic ureolytic CaCO₃ precipitation in a finer grained material. The specimen was prepared in the same manner as described for the triaxial columns for the Ottawa 20-30 sand. The cementation fluid for the first of the two applications contained approximately 2.0 g/L enzyme, 400 mM urea (reagent grade, Sigma-Aldrich), 300 mM CaCl₂-2H₂O (laboratory grade, BDH) at pH=7.7. The fluid for the second application contained 1 M urea-CaCl₂-2H₂O solution at pH=7.8 without any enzyme. After the test, the triaxial specimen was washed and subject to acid digestion in the same manner as the Ottawa 20-30 triaxial specimens.

Results Acrylic Tubes

Approximately 100 ml of cementation solution was delivered per application for the first application in each acrylic tube. However, the amount of solution the tube would accept was notably reduced in subsequent applications, when less than 75 ml was typically required to fill the tubes to z ½ inch (12 mm) above soil line. At the conclusion of the experiment, precipitation was visible along the entire length of tubes 1 and 2. Internally the cementation was variable, with some highly cemented zones and other zones with little to no cementation.

Tube 1 yielded mostly small, loose chunks of sand with strong effervescence upon digestion. Most of this column appeared un-cemented and exhibited unusually viscous behavior when wet. A fairly large (compared to column diameter) piece of strongly cemented sand (not breakable without tools) formed in the deepest layer of tube 1. Tube 2 had many small chunks of weakly cemented sand with strong effervescence upon digestion. Tube 3 had little to no precipitation in the top layer (i.e. this layer did not show any indication of carbonate upon acid digestion.) The deepest layer of tube 3 contained many pieces of weakly cemented sand that effervesced strongly upon digestion. The middle layer of tube 3 contained a few pieces of cemented sand that effervesced moderately upon digestion. The results from the acid washing are presented in Table 1.

TABLE 1 Results from Experiment Set 1 using 20-30 Ottawa silica sand Summary of Results Weight Amt. of Total Amt. Theor. Max Tube Change via CaCO₃ CaCO₃ CaCO₃ # Layer Digestion (g) (g) (g) 1 1  11% 3.57 11.8 ≈14.5 2 3.8% 1.67 3 2.7% 1.73 4 2.1% 1.40 5 2.3% 1.74 6 2.0% 1.64 2 1 0.76%  0.63 2.07 ≈4.35 2 0.65%  0.69 3 0.49%  0.75 3 1 0.23%  0.31 3.57 ≈4.35 2 0.58%  0.63 3 1.7% 2.63

The theoretical maximum CaCO₃ content is the stoichiometric maximum balanced on initial concentrations. The primary experimental differences between the tests are (1) the number of applications of cementation fluid and (2) the manner in which the urease was delivered. The results indicate that there is greater carbonate precipitation with increasing number of applications, as expected. The data show more precipitation in (or on) the top layer of tubes 1 and 2 but not in tube 3, as the enzyme was physically confined to the lower-third layer in tube 3 during sample preparation. In the top layer of tube 3, where no urease was mixed with the sand, carbonate precipitation was nearly undetectable. There was no visual evidence of precipitation and practically no measurable change in weight of this layer after acidification (weight change=0.23%). In the bottom layer of tube 3, where 3 g of dry enzyme was mixed with the soil, there was a weight change of 1.7% following acid washing. The middle layer of this specimen had a minor change in weight (0.58%), possibly due to uneven distribution of the layers during preparation or splitting of the specimen or to upward migration of urease from the bottom layer. XRD analysis confirms that calcite is the mineral phase present in the cemented soil chunks. LV-SEM images, presented in FIG. 1, show silica (quartz) sand particles cemented with calcium carbonate and various morphological features associated with the cementation process on the silica surface.

Triaxial Columns

The three triaxial sand columns (2 Ottawa 20-30 sand columns and 1 Ottawa F-60 sand column) were tested in drained triaxial compression prior to acid digestion. All three columns were able to stand upright after removal of the split mold. The results of the triaxial compression tests performed on the 20-30 Ottawa sand are presented in FIG. 2 and the results for the F-60 Ottawa sand are presented in FIG. 4. The carbonate cement content for one of the 20-30 silica sand columns was 2.0% CaCO₃ (by weight). The carbonate content of the other 20-30 Ottawa sand column could not be quantified due to unintended sample loss. The carbonate cement content for the finer grained F-60 Ottawa sand was 1.6% CaCO₃ (by weight). The results show substantial strength increase for all 3 sand columns tested.

Conclusion

Sand column tests have shown that agriculturally-derived urease can be used to induce calcium carbonate precipitation in sand. Sand columns were developed using Ottawa 20-30 and F-60 sand and three different preparation methods: dry pluviation followed by percolation of a calcium-urease-urea cementation solution, pluviation into a calcium-urease-urea cementation solution, and mixing the sand with urease prior to pluviation with a calcium-urea solution. Cementation was observed in all of the columns. XRD and SEM testing confirmed that calcium carbonate (specifically calcite) was the cementing agent. Acid digestion showed that increased applications yielded correspondingly greater carbonate precipitation. The quality of cementation, as determined by the effort needed to break apart cemented chunks of sand, varied depending on the sampling location within the column. Triaxial test results on cemented columns showed substantial strength increase over non-cemented columns at the same relative density.

Example 2 Methods:

Three sand columns were constructed in 12″ long by 4″ (I.D.) clear PVC tubes (schedule 40) and labeled “Column #1,” “Column #2,” and “Column #3.” Column #1 and Column #2 used Ottawa 20-30 sand, a medium (grain size) sand, while Column #3 used Ottawa F60 sand, a fine sand. Each PVC column was closed-off on one end using a flexible cap (Qwik Cap) and fastened with a hose clamp. The 3 columns were filled with densified sand to depth of approximately 4″. Next, an injection tube was made of flexible ¼″ (O.D.) Tygon tubing with 6-8 needle size holes (18 gauge) in a radial pattern along the last 1.5″ of the tubing. The injection tube was then placed along the axis of the column with the end containing needle holes approximately 0.25″ above the 4″ densified soil layer. The columns were filled with the designated sand using a small scoop to a height of 10″ (i.e., 6″ above the 4″ base layer). Each column was then filled through the injection tube with approximately 700 mL of tap water at 35° C. in order to fully hydrate the columns—the final water level was just above the top of the soil (i.e. slightly more than 10″ from the bottom of the tube). In order to create a uniform soil column, the columns were than densified by firmly tapping the outside of the PVC tube in a radial pattern using a blunt instrument, which reduced the final soil column height from 10″ to approximately 9.4″ in each column. Approximately 40 mL of bentonite slurry was injected into Column #2 through the Tygon injection tube. Each column then received 155 mL of the reaction medium consisting of tap water with 1.36 M urea and 0.765 M calcium chloride dehydrate (pH=7.3, 35° C.). After receiving the reaction medium, the injection tubes were flushed with 2 mL water. Then 15-20 mL of enzyme solution consisting of 0.44 g/L urease enzyme (Sigma Aldrich Type-III, Jack Bean Urease, 26,100 U/g activity) and 4 g/L stabilizer (nonfat powdered milk) were introduced to each column through the injection tube. Finally, another 3-4 mL of reaction medium was injected into each column followed by a 2 mL water flush. The injection tubes were closed with polypropylene pinch clamps and the columns were capped with plastic clear wrap and placed in dark, warm (30° C.) environment for 28 days. On day 7, an additional 115 mL of reaction medium and 10 mL of enzyme solution was delivered to each column in a manner similar to described above. Approximately 3 ml (0.1% v/v) of 1M NaOH was injected into each column 48 hours before the experiment terminated.

Results:

Upon disassembly, all 3 columns showed strong carbonate cementation in a cylindrical zone around the end of the inject tube. The results varied slightly for each column, as follows:

Column #1 (20-30 medium sand)—A region of strongly cemented soil began ≈2.5″ from the column bottom. The cemented zone was ≈4.5″ in length and displayed a prominent rounding at the top and a flat surface at the bottom. Overall, the cemented region appeared to have a cylindrical bottom and bulb-shaped upper portion (FIG. 4). Several chunks of cemented soil were dislodged to access the most strongly cemented region which had the injection tube firmly embedded. The cemented region could not be dislodged from the PVC column without the use of hand tools. Column #2 (20-30 medium sand w/40 mL bentonite slurry)—A cylindrical region of strongly cemented soil began ≈3″ from the column bottom, was ≈4″ in length and displayed small rounding near the top and a flat surface at the bottom. Overall, the cemented region was mostly cylindrical (FIG. 5). Several chunks of cemented soil were dislodged to access the most strongly cemented region which had the injection tube loosely embedded. The cemented region could not be dislodged from the PVC column without the use of hand tools. Column #3 (F-60 fine sand)—A region of strongly cemented soil began ≈3″ from the column bottom, was ≈2.5″ in length and displayed a clear bell-shaped top and a flat surface at the bottom. Overall, the cemented region appeared bell-shaped (FIG. 6). The entire soil column was dislodged upon disassembly and many chunks of cemented soil were dislodged to access the most strongly cemented region which had the injection tube firmly embedded. In addition, the soil overall mass contained many small (1-3 mm) pieces of cemented sand. The column had a strong smell of ammonia and displayed significant amounts of gas bubbles during washing.

Example 2 demonstrates (for example) the ability to create cylindrical columns in a saturated soil (i.e. below the water table); stabilize a fine grained material; and the optional facilitation of cementation by first injecting a bentonite slurry into the column, which provides particular benefit when used with more porous (coarser) starting materials. 

What is claimed is:
 1. A mineral precipitation method, comprising: combining a saturated, porous, particulate starting material with (a) isolated urease; (b) urea; and (c) a source of divalent cations; wherein (a), (b), (and (c) are provided in amounts effective and the combining is carried out under conditions suitable to cause carbonate precipitation of the starting material.
 2. The method of claim 1, further comprising combining the starting material with powdered milk to act as a stabilizer.
 3. The method of claim 1, wherein the method is used for one or more of improving bearing capacity of foundations; reducing settlement potential of foundations or embankments; stabilizing slopes; reducing the potential for earthquake-induced liquefaction; mitigating the potential for damaging ground displacements subsequent to earthquake-induced liquefaction; increasing lateral resistance of foundations; enhancing stability of slopes or embankments; reducing lateral earth pressures on retaining walls; increasing passive resistance of retaining walls; increasing capacity of ground anchors or soil nails; increasing the side resistance and tip resistance of deep foundations; facilitating tunneling in running or flowing ground; stabilizing excavations bottoms; soil erosion control; or groundwater control.
 4. The method of claim 1, wherein the starting material comprises one or more of sand, silt, clay, sawdust, igneous rocks, metamorphic rocks, gravel, fractured crystalline rocks, cracked concrete, or sedimentary rocks.
 5. The method of claim 1, wherein the source of divalent cations comprises a source of divalent calcium ions.
 6. The method of claim 1, wherein the combining step is carried out more than once.
 7. The method of claim 1, wherein the combining comprises: (i) mixing the urease with the starting material; and (ii) injecting into the starting material, under pressure and via an injection tube, a solution comprising the urea and the source of divalent cations.
 8. The method of claim 7, wherein the injecting is carried out two or more times.
 9. The method of claim 7, wherein the urease is mixed with starting material in only a portion of the starting material prior to the injecting.
 10. The method of claim 1, wherein the method further comprises introducing a clay slurry into the starting material prior to or concurrent with combining the starting material with the isolated urease, the urea, and the source of divalent cations.
 11. The method of claim 1, wherein the saturated starting material comprises a column of the saturated starting material.
 12. The method of claim 1, wherein the combining is conducted underneath or adjacent to an existing structure that is sensitive to ground settlement or heave.
 13. The method of claim 12, wherein the carbonate precipitation does not cause ground settlement or heave affecting the existing structure. 